Expression and Localisation of WHY1 in Wild type and WHY knockdown Barley Leaves
Since monocotyledonous leaves show a gradient of development from base to tip, we sampled the base and tip of the first leaves of 7-day old wild type seedlings to establish the intracellular localisation of WHY1. The levels of WHY1 transcripts were highest in the basal regions of 7-day old wild type leaves decreasing progressively from the middle to tip (Fig. 1). Immunogold labelling revealed that the WHY1 protein was found in developing plastids of the cells in the lowest region of the leaf bases that are not exposed to light (Fig. 2A), as well as in mature chloroplasts in the leaf tip (Fig. 2B). Furthermore, imaging of the nucleus in the leaf base revealed that gold labelling was abundant (Fig 2C, D) where it appeared to be mostly clustered with electron dense chromatin (Fig. S1). In the leaf base we observed an average density of 15.88 ± 2.04 (n = 8) gold particles in plastids and 26.30 ± 2.40 (n = 10) gold particles in nuclei however, due to the presence of large vacuoles, we were unable to obtain high quality nuclear images from the leaf tip. Labelling was largely absent from the cytosol (Fig. 2).
WHY1-deficient barley leaves develop in a similar manner to the wild type except that the greening of each leaf is delayed in the absence of WHY1 (Krupinska et al., 2020). To obtain an understanding of developmental delay we divided leaves into three sections; base, middle and tip. The basal sections of 7-day old wild type barley leaves had significantly less chlorophyll (Fig. S2) than the middle and tip sections. A similar chlorophyll gradient was observed in W1-1 and W1-7 leaves although the chlorophyll content in each section was lower than in the corresponding wild type leaves (Fig. S2).